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Generation of 3D Tumor Spheroids for Drug Evaluation Studies
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Automated High-Content, High-Throughput Spatial Analysis Pipeline for Drug Screening in 3D Tumor Spheroid Inverted

Hyunsu Jeon1, Gaeun Kim1, James Carpenter1

  • 1Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States.

ACS Applied Materials & Interfaces
|August 9, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces a 3D high-content, high-throughput screening platform using tumor spheroid arrays for efficient drug discovery. The novel system offers rapid, spatially resolved therapeutic profiling and enhanced analysis for 3D models.

Keywords:
automated processingcancer modeldrug discoveryhigh yieldhydrogelspatial signal profiling

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Area of Science:

  • Biomedical Engineering
  • Drug Discovery
  • Cancer Research

Background:

  • Traditional 2D assays lack physiological relevance for drug screening.
  • Current 3D spheroid systems face scalability, uniformity, and analytical efficiency challenges.
  • Need for advanced platforms for robust 3D drug screening.

Purpose of the Study:

  • To develop a fully integrated 3D high-content, high-throughput (HCHT) screening platform.
  • To enable rapid and spatially resolved therapeutic profiling using tumor spheroid arrays.
  • To overcome limitations of existing 3D screening methods.

Main Methods:

  • Utilized a bioinert inverted colloidal crystal (iCC) hydrogel framework for tumor spheroid array generation.
  • Integrated an automated, high-speed image analysis pipeline for rapid data processing.
  • Employed fluorescence-weighted-spheroid centroiding (FwSC) for multiparametric spatial analysis.

Main Results:

  • Achieved high spheroid density (∼79.8 spheroids·mm-2) with tight size uniformity (<10% SD).
  • Automated image analysis processed images in <5 s, significantly faster than conventional methods.
  • Captured dose-dependent drug diffusion, extracellular vesicle penetration, and immune cell infiltration dynamics.
  • Revealed distinct spatial toxicity signatures for chemotherapeutics, emphasizing spatial context.

Conclusions:

  • The developed platform enables rapid, reproducible, and spatially informative 3D screening.
  • Offers a powerful tool for drug discovery, tumor modeling, and immunotherapeutic development.
  • Highlights the critical role of spatial context in assessing drug response.